CN105613302A

CN105613302A - Method for sweet potato stem tip in vitro rapid propagation

Info

Publication number: CN105613302A
Application number: CN201610178808.8A
Authority: CN
Inventors: 孙小芹; 李定红; 杭悦宇
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2016-03-28
Filing date: 2016-03-28
Publication date: 2016-06-01

Abstract

The invention discloses a method for sweet potato stem tip in vitro rapid propagation. Through in vitro culture of mature and delicate stem tips of sweet potatoes, the optional experimental conditions are screened, the optional rapid propagation system for sweet potato culture is established, and the fact that the optimal hormone concentration in budding inducing culture medium is NAA 0.1 mg/L plus KT 2.0 mg/L, the optimal hormone concentration in subculture medium is NAA 0.5 mg/L plus KT 2.0mg/L, and the optimal hormone concentration for rooting culture is IBA 2.0mg/L can be obtained. The method can rapidly obtain virus-free seedlings of sweet potatoes, improves the quality and yield of sweet potatoes, and provides a theoretical basis and practice guidance for the industrialization production of sweet potatoes.

Description

A kind of sharp method bred fast from body of yam stem

Technical field

The invention belongs to field of plant tissue culture, it relates to a kind of sharp method bred fast from body of yam stem.

Background technology

Yam (D.esculenta (Lout) brukill) belongs to food plant not of the same race together for Chinese yam, Chinese yam is Dioscoreaceae (Dioscoreaceae) Wild yam (DioscoreaL.) Chinese yam (D.polystachyaTurcz.), just it is listed in from " Shennong Bencaojing " and top grade namely nourishes food, containing polysaccharide, starch, protein, the multiple compositions such as total free aminoacids and trace element, there is immunomodulatory, anti-oxidant, hypoglycemic, reducing blood-fat, antitumor, anti-mutation, regulate the effects such as spleen and stomach function, it it is the traditional tonic medicine of Chinese Famous, also it is traditional healthcare dish. the edible part of yam is the spherical stem tuber of similar potato-like, and fine and tender taste, food flavor are good, entrance is soft, can do vegetables, coarse cereals or medicinal etc.

Current yam is farmers''s microspecies that are wide in variety but that cultivate without scale, we introduce a fine variety from Hainan farmers' in recent years, and obtained by winter hardiness screening and fit raw strain in Jiangsu, being found by nutritive ingredient detection, its polysaccharide content is 3 ~ 4 times of the highest iron rod yam of polysaccharide content that existing market is generally acknowledged; And the taproot class yam variety of cultivation main from Jiangsu is different, yam is shallow class plant, and plantation is without the need to digging zanjon, build high ridge, sleeve pipe etc., and results are also easy, therefore, significantly reduce production cost; Yam plant can grow 30-40 spherical tuber, and per mu yield can reach more than 3000 jin, and output is big; Simultaneously, with regard to its social benefit, the resistance to fertilizer of yam drought resisting, few disease and pest and growing way are strong, it is adapted at the low mountains and hills area plantation of Jiangsu Province's lack of water fertilizer deficiency, and because it is less demanding to illumination, not only can be planted on existing arable land, it is also possible to the vacant lot plantation between secondary forest, economic forest, land resources need not be seized with existing farm crop, thus alleviate the contradiction that land resources is day by day deficient. Polysaccharide content height, shallow root system have easily planted the plurality of advantages such as receipts, output height, the suitable life in Jiangsu so that yam becomes Chinese yam that one emerging, that potentiality to be exploited is big, prospect is good and newly plants. Be not only southern Jiangsu even revive in the area Chinese yam producer provide Chinese yam best in quality and newly plant, and enriched the vegetables market of human consumer, be more conducive to the renovation and utilization in low mountains and hills area, Jiangsu, there is good economical effectiveness and social benefit.

In addition, traditional yam planting be healthy and strong in harvest season choosing, without the stem tuber storage of disease and pest, taking out planting time next year and cut off plantation, there is the problems such as kind of block serious waste, reproduction speed are very slow, manpower and materials consumption is big in this kind of traditional " spring sowing, autumn harvest, winter hide " mode; And, due to long-term vegetative propagation, cause accumulation and the propagation of virus and disease, cause quality deterioration, output to decline. Forefathers' research shows, it is possible not only to greatly improve Chinese yam breeding coefficient by the method for tissue culture Virusfree, and the problems such as the long-term nutrition virus disease that causes of breeding and quality deterioration can be solved, existing a lot of report in a variety of or kind is such as iron rod yam, RHIIZOMA DIOSCOREAE from Henan of China, fork stamen Chinese yam etc. Yam there is not yet the research report that tissue culture obtains detoxic seedling, the present invention will be research object taking yam, by multifactor multilevel experiments such as different explants, hormon kind and concentration process, filter out optimum experimental condition, establish the best rapid propagation system of yam tissue culture, for the industrialization production of yam provides theoretical foundation and practical advice.

Summary of the invention

The present invention provides a kind of sharp method bred fast from body of yam stem, pass through tissue culture technique, select yam stem point, inoculate after sterilization and cultivate into the substratum of different hormones and concentration, and filter out the best system obtaining detoxic seedling fast, solve the problem of the long-term vegetative propagation quality deterioration of yam, output decline.

In order to achieve the above object, the technological step that the present invention adopts is as follows:

(1) the young tender stem point being adopted healthy yam is carried out sterilization;

(2) the stem point after sterilization in step (1) is cut wound part;

(3) the stem point in step (2) is accessed in the MS substratum of sterilizing and carry out inducing Buds formation Multi bud body;

(4) succeeding transfer culture is carried out by proceeding to subculture medium after the Multi bud body plant division in step (3);

(5) what grown into by subculture in step (4) proceeds to root media without root seedling and carries out root culture.

Wherein the point of the young tender stem in step (1) can adopt the stem point that yam grows about plant after three months;

In step (2), stem point cuts the wound contacted with thimerosal;

In step (4), Multi bud body wants plant division succeeding transfer culture;

In step (5), the base portion of seedling must insert and grow in substratum.

Operation in step (2) (3) (4) (5) is best to be completed on Bechtop, operates under will remaining on aseptic condition;

The present invention provides the best group training system obtaining yam detoxic seedling and breeding fast, to improve the quality and yield of yam, has bigger actual application value.

Accompanying drawing explanation

Fig. 1 is that stem point has just started to access substratum.

Fig. 2 is that stem tip culture has gone out Multi bud body.

Fig. 3 accesses substratum after Multi bud body plant division.

Fig. 4 accesses, without root little Miao, the detoxic seedling that root media grows root by what turn out.

Embodiment

In following embodiment, method therefor is if no special instructions, is ordinary method. MS substratum, sucrose, agar, hormone KT(kinetin) produce by Suo Laibao company, 1/2MS substratum is produced by Shanghai Li Chen commerce and trade company limited, alcohol is produced by Nanjing chemical reagent company limited, PVP K-30 (PVP) is produced by Chemical Reagent Co., Ltd., Sinopharm Group, hormone NAA(a-naphthylacetic acid) and IBA(3-indolebutyric acid) produce by Shanghai Kai Yang Bioisystech Co., Ltd, hormone 6-BA(6-benzyl aminoadenine) purchased from Rui Yong bio tech ltd, Shanghai, in experiment, yam is planted in Nanjing Botanical Garden Mem. Sun Yat-Sen. below its culturing process is described in detail.

1. sample

The yam introduced a fine variety in Nanjing Botanical Garden Mem. Sun Yat-Sen garden gets growth is strong, the young tender stem point of anosis worm harm plant, clean with tap water, by rinsed with sterile water 3��5 times.

2. the preparation of substratum

Inducing culture: adopt MS minimum medium, add sucrose 30g/L and agar 10g/L, the NAA (0.1mg/L of additional different concns, 0.5mg/L)+KT (1.0mg/L, 2.0mg/L) on the basis of inducing culture, suitably adjust, do subculture medium.

Subculture medium: adopt MS minimum medium, add sucrose 30g/L and agar 10g/L, 0.5mg/L+KT2.0mg/L.

Root media: adopt 1/2MS substratum, add sucrose 30g/L and agar 10g/L, IBA2.0mg/L, is adjusted to 5.8 by the pH value of substratum before sterilizing, autoclaving (121 DEG C) 20min after sealing.

Above substratum all adds PVP0.01%, for subsequent use after cooled and solidified.

3. explant sterilization

On Bechtop, by clean stem point with 75% ethanol disinfection 10-15s, then put into 0.1%HgCl2 solution and sterilize 3-4min, after sterilization, with aseptic water washing 4-5 time.

4. inoculate explant on inducing culture

The stem point that explant after sterilization is cut into long about 0.5cm, is seeded on inducing culture, and each process 10 bottles inoculates 3 for every bottle.

5. inducing culture

Culturing room is put into, temperature (24 �� 2) DEG C, illumination every day 16h, intensity of illumination 2000lx after cultivating inoculation. Routine observation record pollution condition, changes a substratum, adds up free of contamination germination rate after 30d for every two weeks. After Multi bud body is formed, plant division succeeding transfer culture.

6. root culture

When bud seedling grows to 2 ~ 3cm, the bud seedling choosing robust growth is inoculated in root media root induction, every bottle of inoculation 3 bud seedlings, and 30 days " Invest, Then Investigate " tissue cultured seedling are taken root situation.

7. observe statistics growth result

In inducing culture, best hormone concentration is NAA0.1mg/L+KT2.0mg/L as can be seen from Table 1, and experiment show that the best hormone concentration of subculture growth medium is NAA0.5mg/L+KT2.0mg/L, and the hormone concentration of root culture the best is IBA2.0mg/L.

Table 1 hormon concentration is on the impact of the induction of the pointed one-tenth Multi bud body of stem

Claims

1. the sharp method bred fast from body of yam stem, it is characterised in that, comprise the steps:

(2) the stem point after sterilization in step (1) is accessed in the MS substratum of sterilizing and induce Multi bud body;

(3) regeneration bud in step (2) is proceeded to subculture medium and carry out succeeding transfer culture;

(4) proceeding to growing in step (3) root media without root seedling and carry out root culture.

2. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, the young tender stem point described in step (1) can adopt yam growth about plant after three months, clean with tap water, by rinsed with sterile water 3��5 times.

3. the sharp method bred fast from body of yam stem according to claim 1, it is characterized in that, the sterilization method described in step (1) is again with 75% ethanol disinfection 10-15s, then puts into 0.1%HgCl2 solution and sterilize 3-4min, after sterilization, with aseptic water washing 4-5 time.

4. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, in step (2), stem point is cut into long about 0.5cm.

5. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, the stem point described in step (2) to be excised the wound of contact mercuric chloride.

6. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, the inducing culture described in step (2) adopts MS minimum medium, adds sucrose 30g/L and agar 10g/L.

7. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, in the inducing culture filtered out described in step (2), best hormone concentration is NAA0.1mg/L+KT2.0mg/L.

8. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, the best hormone concentration of subculture medium described in step (3) is NAA0.5mg/L+KT2.0mg/L, identical described in other compositions of substratum and claim 6.

9. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, the hormone concentration of the root culture the best described in step (4) is IBA2.0mg/L, identical described in other compositions of substratum and claim 6.

10. the sharp method bred fast from body of yam stem according to claim 1, it is characterised in that, described in step (4) when being grown to 2 ~ 3cm without root seedling, that chooses robust growth is inoculated in root media root induction without offspring, every bottle of inoculation 3.

The 11. sharp methods bred fast from body of yam stem according to claim 1, it is characterised in that, the substratum described in step (2) (3) (4) all adds PVP0.01%.

The 12. sharp methods bred fast from body of yam stem according to claim 1, it is characterised in that, the culture condition described in step (2) (3) (4) is all put into culturing room, temperature (24 �� 2) DEG C, illumination every day 16h, intensity of illumination 2000lx.